To identify the upstream events controlling the regulation of flooding-responsive proteins in soybean, nuclear proteomic analysis in root tip was performed. A total of 365 nuclear proteins were significantly changed in the root tip of soybean during the initial stage of flooding stress. Four exon-junction complex-related proteins, which are involved in mRNA transport, and NOP1/NOP56, which function upstream of pre-60S ribosome biogenesis, were increased and decreased in untreated and flooded soybean, respectively. Proteomic analysis of total proteins revealed that the function of cytosolic ribosomes was suppressed by continuous flooding stress. Protein-protein interaction analysis indicated that 17 chromatin structure-related proteins were decreased in soybean root tip in response to flooding stress and that these proteins formed an interacting network. Among 15 examined histone proteins, histone H3 was clearly decreased during the initial stages of flooding stress. Additionally, a number of protein synthesis-, RNA-, and DNA- related nuclear proteins were significantly changed in a time-dependent manner. mRNA expression analysis demonstrated that the genes encoding the significantly changed flooding-responsive nuclear proteins were inhibited by the transcriptional inhibitor actinomycin D. These results suggest that ribosome function may be suppressed in flooding-stressed soybean through the activities of pre-ribosome biogenesis- and mRNA processing-related proteins stored in nuclei. In addition, flooding stress may induce changes in chromatin structure and gene expression through the regulation of histone variants in soybean root tip under initial flooding stress.